Nitric oxide (NO) signaling is essential to several physiological functions, and dysfunction in the in this signaling cascade is implicated in multiple diseases such as erectile dysfunction, heart disease, neurodegeneration, stroke, hypertension, and gastrointestinal disease. Soluble guanylate cyclase (sGC) is the primary receptor for NO. NO regulates sGC at two levels and this is consistent with pharmacological observations of NO signaling that are consistent with a two-step activation mechanism by NO. The amplitude and duration of these effects of NO in neuronal signaling, cardiac function, vascular tone and vasodilation are vital to proper function, but the mechanism for the two-step activation by NO has not been thoroughly investigated. A new paradigm for NO signaling through sGC has emerged. Understanding how sGC switches from a low to high activation state is central to this new paradigm. In addition, sGC has become a therapeutic target for the treatment of two forms of pulmonary hypertension: chronic thromboembolic pulmonary hypertension and pulmonary arterial hypertension with the FDA approved Adempas. Our specific aims include: (i) How does NO activate sGC?, (ii) What is the structural architecture of full-length sGC and what are the inter-domain interactions that contribute to the activation mechanism of sGC?, and (iii) What is the mechanism of action of Adempas (riociguat) and related stimulators of sGC. Experimental approaches will include the following biochemical methods: enzyme kinetics, cloning, expression, purification and characterization of wild type and site-directed mutants of sGC, electron microscopy structural methods, hydrogen-deuterium exchange and peptide mapping. It is a central goal of this proposal to develop a complete molecular level view of the complex relationship between NO, drugs like Adempas and sGC. The extension of this work into physiological function will provide a rational basis for the understanding and treatment of NO signaling disorders in human disease.